Mechanisms That Limit the in Vitro Proliferative Potential of Human CD8 + T Lymphocytes

Mechanisms That Limit the In Vitro Proliferative Potential of Human CD8 + T Lymphocytes

This information is current as Marco Migliaccio, Kenneth Raj, Olivier Menzel and of September 27, 2021. Nathalie Rufer J Immunol 2005; 174:3335-3343; ; doi: 10.4049/jimmunol.174.6.3335 http://www.jimmunol.org/content/174/6/3335 Downloaded from

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2005 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Mechanisms That Limit the In Vitro Proliferative Potential of Human CD8؉ T Lymphocytes1

Marco Migliaccio,* Kenneth Raj,† Olivier Menzel,* and Nathalie Rufer2*

Human T lymphocytes can be numerically expanded in vitro only to a limited extent. The cyclin-dependent kinase inhibitor p16INK4a is essential in the control of cellular proliferation, and its expression, in epithelial cells, is associated with irreversible growth arrest. Using long-term cultured CD8؉ T lymphocytes, we have investigated the role of the p16/pRb pathway in the regulation of T cell proliferation and senescence. In this study, we describe at least two mechanisms that cause replicative growth arrest in cultured lymphocytes. The ﬁrst one depends on the expression of p16INK4a and is directly responsible for the exit of a signiﬁcant proportion of CD8؉ T cells from the proliferative population. This induced p16 expression pattern is observed during each round of mitogen stimulation and is not related to activation-induced cell death. Importantly, knocking down p16INK4a

expression allows increased proliferation of T cells. The second one is a phenomenon that resembles human ﬁbroblast senescence, Downloaded from but is independent of p16INK4a and of telomere attrition. Interestingly, virtually all pRb proteins in the senescent population are found in the active form. Our data indicate that newly synthesized p16INK4a limits the proliferation of T lymphocytes that respond to mitogen, but is not required for the loss of mitogen responsiveness called senescence. The Journal of Immunology, 2005, 174: 3335–3343.

ost human T lymphocytes isolated from peripheral fore, to enter the cell division cycle, proteins of the pRb family, http://www.jimmunol.org/ blood or other sources are in a quiescent state. They especially pRb itself, must be inactivated, by cyclin-dependent ki- M can be stimulated to proliferate in culture by mito- nases (CDKs),3 which phosphorylate pRb (reviewed in Refs. 4 and gens and IL-2. After such stimulation, the cells undergo a num- 5). Together, the core members of the pRb pathway (pRb, E2F, ber of divisions before they re-enter quiescence, which can and cyclin CDKs) regulate the progression of cells into the cell again be overcome by repeating the stimulation procedure. Af- division cycle. ter a number of restimulations, T lymphocytes cease to respond. The factors that cause cultured T lymphocytes to cease respond- This state, termed replicative senescence, appears to be irre- ing to stimulation and enter a state of irreversible growth arrest versible even though the cells remain alive so long as IL-2 is have not been clearly identified. In mesenchymal and epithelial periodically added (reviewed in Ref. 1). Replicative senescence cells grown under optimal culture conditions, replicative senes- by guest on September 27, 2021 imposes a limit to the numerical expansion of human T cells in cence involves telomere shortening. In most human cell types, vitro, which constitutes a major barrier in the development of some telomere repeats are lost during each cell division. Cells in adoptive transfer approaches based on the use of Ag-specific which one or more telomeres have become too short to prevent the cytolytic CD8ϩ T lymphocytes (CTLs), in which substantial chromosome end from being treated as a double strand break un- numbers of T cells must be generated in vitro (2). dergo a proliferation arrest mediated by the p53 and pRb pathways Many studies have focused on the identification of the molecular (6). In various primary human cell types, the causal link between mechanisms that trigger cell cycle entry, progression, and exit, telomere shortening and cellular senescence has been established thereby regulating lymphocyte proliferation and their numerical by showing that transduction of the human telomerase reverse- expansion. The E2F transcription factors are required for T lym- transcriptase (hTERT) gene results in extension of telomeres and phocytes to enter the cell division cycle. E2Fs play a pivotal role an increase in the proliferative life span (7, 8). Apart from criti- in the timely activation of a number of genes coding for proteins cally short telomeres, tissue culture conditions that inadvertently that participate in DNA synthesis (reviewed in Ref. 3). The pRb stress cells may also lead to the irreversible growth arrest observed family proteins (pRb, p107, and p130) prevent cellular division by in in vitro cultures. This has been most clearly shown in epithelial binding to and inhibiting the transcriptional activity of E2F. There- cells, which stop to proliferate even though the lengths of their telomeres are well beyond the critical limit (9, 10). In some cell types, the presence of the p16INK4a (hereafter p16) *Swiss Institute for Experimental Cancer Research, National Center of Competence in Research Molecular Oncology, Epalinges, Switzerland; and †National Institute for protein is expressed in response to various forms of cell stress and Medical Research, The Ridgeway, London, United Kingdom is associated with irreversible growth arrest (11–13). High levels Received for publication July 20, 2004. Accepted for publication December 17, 2004. of p16 protein are detected in keratinocytes that senesce despite The costs of publication of this article were defrayed in part by the payment of page having long telomeres (9, 10, 14, 15). High levels of p16 have also charges. This article must therefore be hereby marked advertisement in accordance been found in senescing human fibroblasts (16–18), and, together with 18 U.S.C. Section 1734 solely to indicate this fact. with the p53 pathway, the p16/pRb pathway has been shown to be 1 M.M. is supported by a grant of the Ligue Suisse contre le Cancer (OncoSuisse; involved in the response to uncapped telomeres in such cells (19). OCS 1228-02-2002). O.M. and N.R. are supported by the National Center of Com- petence in Research Molecular Oncology, a research instrument of the Swiss National Thus, p16 can mediate senescence induced either by critically Science Foundation. 2 Address correspondence and reprint requests to Dr. Nathalie Rufer, Swiss Institute for Experimental Cancer Research, National Center of Competence in Research Mo- 3 Abbreviations used in this paper: CDK, cyclin-dependent kinase; 7-AAD, 7-ami- lecular Oncology, 155 ch. des Boveresses, CH-1066 Epalinges, Switzerland. E-mail noactinomycin D; FISH, fluorescence in situ hybridization; hTERT, human telomer- address: [email protected] ase reverse transcriptase; PD, population doubling; shRNA, short hairpin RNA.

Copyright © 2005 by The American Association of Immunologists, Inc. 0022-1767/05/$02.00 3336 p16INK4a EXPRESSION IN CULTURED HUMAN CD8ϩ T LYMPHOCYTES short telomeres or inadequate culture conditions. The p16 protein Aldrich), and 50 ␮l of cell suspension was mixed with 5 ␮l of PE-conju- is a CDK inhibitor that prevents phosphorylation and inactivation gated mAbs against human p16, pRb, hypophosphorylated, active form of of the pRb protein by cyclin D-Cdk4/6 (20) and inhibits the tran- pRb, or corresponding isotype controls (BD Biosciences). Cells were in- cubated1hatroom temperature, washed with permeabilization solution, sition of cells from the G0 to the G1 phase of the cell cycle (21). resuspended in 400 ␮l of PBS/3% FCS, and kept at 4°C until analyzed on The importance of p16 in normal cell proliferation is reflected in a FACSCalibur flow cytometer (BD Biosciences). Early apoptotic cells the high frequency of mutations that inactivate the function of p16 (annexin V-positive) and allogeneic PBMC cells (CFSE-negative) were in numerous types of human cancers, including the majority of T discarded by gating the cells in the corresponding plots. For BrdU incorporation studies, 106 cell samples were incubated in the presence of 10 cell leukemias (22, 23). Recent analysis of p16-deficient mice ␮g/ml BrdU during1hat37°C. Cells were then fixed, permeabilized, demonstrated the importance of p16 in restraining the incidence treated with DNase to expose BrdU epitopes, and stained with an anti- and development of tumors (24). BrdU Ab and 7-aminoactinomycin D (7-AAD) using the BrdU flow kit At present, there is no consensus as to why cultured T lympho- (BD Biosciences). cytes cease to proliferate after a certain number of stimulation Data were expressed as: 1) normalized mean fluorescence (Figs. 2 and 7) or as 2) percentage of p16-positive cells (Figs. 3–6). 1) Histograms cycles. Proliferation arrest could be due to inadequate culture con- obtained from cells stained with each Ab and the corresponding isotype ditions, or to telomere shortening. In either case, we expect an control were generated. The normalized mean fluorescence for every mAb involvement of p16. We have assessed the status of the p16/pRb in respect to its isotype control was first calculated using the geometric ϩ pathway in human CD8 T lymphocytes that were subjected to mean values of each histogram (Xmab and Xiso), according to the following Ϫ formula: ((Xmab Xiso)/Xiso). To facilitate the comparison between sam- repeated rounds of mitogenic stimulation until replicative growth ples, these mean fluorescence values were then normalized with the mean arrest. We observed that p16 expression was directly induced as a fluorescence value obtained from fresh cells that did not proliferate at day consequence of T cell activation. A large fraction of T cells ex- 4 after stimulation (CFSEbright). This normalization was arbitrarily set to 1 Downloaded from pressed high levels of p16 in response to each round of stimulation (total pRb and active pRb) or 10 (p16) and expressed as normalized mean and was not related to cells that underwent activation-induced cell fluorescence. 2) For quantifying the percentage of p16-positive cells (%), histograms obtained with anti-p16 or isotype control were overlaid. In death. Under the culture conditions used, replicative senescence of ϩ these p16 histograms, the area that overlapped the isotype histogram rep- CD8 T cells was observed after 30 population doublings (PD), resented the p16-negative area (area p16Ϫ), while the area that did not but curiously, it did not correlate with high p16 levels or telomere overlap the isotype histogram represented the p16-positive area (area p16ϩ). Both areas were determined using Adobe Photoshop software attrition. Taken together, our results indicate that the in vitro rep- http://www.jimmunol.org/ (Adobe Systems), and the percentage of p16-positive cells was calculated licative potential of mitogen-activated human T lymphocytes is as%ofp16ϩ ϭ (area p16ϩ)/((area p16ϩ) ϩ (area p16Ϫ)) ϫ 100. The limited by at least two independent mechanisms, neither of which specificity of the anti-human p16 mAb was confirmed using HeLa (p16ϩ) is associated with telomere shortening, and only one of which is and U2OS (p16Ϫ) cell lines (American Type Culture Collection). associated with p16 expression. Knockdown of p16 expression with specific short hairpin RNA Materials and Methods (shRNA) oligonucleotides ϩ Long-term culture of human CD8 T lymphocytes Small interfering RNAs directed against two target sequences of the first exon of INK4a (p16shRNA-A (GAGGAGGTGCGGGCGCTGC) and

PBMC were obtained from healthy donors by density centrifugation on by guest on September 27, 2021 ϩ p16shRNA-B (GTGCTCGGAGTTAATAGCA)) have been previously re- Ficoll-Hypaque (Pharmacia), and CD8 T lymphocytes were purified by ported to knock down p16 gene expression (26, 27), and were thereby magnetic sorting using a miniMACS device (Miltenyi Biotec). Bulk cul- chosen to create two lentiviral vectors. Oligonucleotides designed to form tures of human T cells were obtained by seeding them onto 24-well plates 6 shRNA targeting these sequences (28) were subcloned in a pSuper vector (10 cells in 2 ml/well) in RPMI 1640 medium supplemented with 8% under the control of the H1 promoter. DNA cassettes containing the H1 human serum and 150 U/ml human rIL-2 (a gift from GlaxoSmithKline) promoter and each of the specific shRNA were then subcloned in a third ␮ ϫ 6 and stimulated with 1 g/ml PHA (Sodiag) plus 0.5 10 /ml irradiated generation lentiviral vector containing the enhanced GFP under the control allogenic PBMC (3000 rad) as feeder cells. Culture medium was checked of the CMV promoter, kindly provided by L. Naldini (Laboratory of Gene daily and changed when required. The stimulation procedure was repeated Transfer and Therapy, Institute for Cancer Research and Treatment, Can- every 10 or 15 days of culture. PDs were determined by counts of cells diolo, Italy). Vesicular stomatitis virus-pseudotyped lentivirus was pro- alive once or twice per week. When indicated, samples of 106 cells were ␮ duced by transfecting 293T cells with the corresponding plasmids, as de- resuspended in 1 ml of CFSE (Molecular Probes) solution (2 g/ml in scribed (29), and 10 ␮l of concentrated viral supernatant (ϳ108 PBS), incubated for 10 min at 37°C, washed twice, and seeded into 24-well transduction U/␮l) was used to infect 106 resting lymphocytes in 0.5 ml of plates, as described (25). complete medium with 1 ␮g/ml polybrene (Sigma-Aldrich). Four to 12 h Western blot analysis after infection, cells were washed and stimulated with PHA and allogeneic feeder cells, as described above. The transduction efficiency typically A total of 5 ϫ 106 cell samples was spun down, and cell extracts were ranged between 30 and 60%. obtained by resuspending the pellets in a lysis buffer (1% SDS, 1 mM sodic orthovanadate, and 10 mM Tris-HCl, pH 7.4). The protein concentration of Long-term culture of hTERT-transduced CD8ϩ T cell clones supernatants recovered after centrifugation was measured by spectropho- ϩ tometry with a protein assay kit (Bio-Rad). A total of 20 ␮l of extract Human CD8 T cell clones expressing either hTERT/GFP or GFP alone corresponding to 8 ϫ 105 cells was loaded in each well, and the proteins were obtained and cultured following serial rounds of subcloning steps, as previously described (30). Briefly, transduced T cells expressing high lev- were resolved on an 8% (for detection of pRb, p130, and p107) or a 12% ϩ (for the other proteins) polyacrylamide gel by SDS-PAGE and electroblot- els of GFP were sorted by a FACStar flow cytometer (BD Biosciences) ted to polyvinylidene difluoride membranes by semidry transfer. The mem- and further recloned by limiting dilution using PHA and feeder cells, as branes were blocked and probed with the indicated Abs: anti-pRb, anti-p16 described for bulk cultures. This subcloning procedure was repeated until (BD Pharmingen), anti-p27kip1, anti-p107, anti-p130, and anti-␣-actin no further subclones could be obtained (GFP control clones) or until the (Santa Cruz Biotechnology). Ab-Ag interaction of the primary Abs was ninth recloning step (hTERT/GFP clones). detected by incubation with secondary HRP-conjugated Abs and subse- quent detection by ECL (Amersham). Telomere fluorescence in situ hybridization (FISH) and flow cytometry (flow FISH) Annexin V staining, intracellular staining, and flow cytometry analysis The average length of telomere repeats at chromosome ends from in vitro cultivated T lymphocytes was measured by FISH and flow cytometry (flow Collected samples of fresh or cultured T lymphocytes were washed and FISH), as previously described (31, 32). Telomere fluorescence was cal- stained with Cy-5-conjugated annexin V (BD Biosciences), according to culated by subtracting the mean fluorescence of the background control (no manufacturer instructions. Cell pellets were then washed, fixed in PBS/2% probe) from the mean fluorescence obtained from cells hybridized with the paraformaldehyde, permeabilized in PBS/3% FCS, 0.5% saponin (Sigma- telomere probe after calibration with FITC-labeled fluorescent beads The Journal of Immunology 3337

(Quantum TM-24 Premixed; Bangs Laboratories) and conversion into mol- designate the growth arrest of diploid human fibroblasts after a ecules of equivalent soluble fluorochrome units. The following equation finite number of cell divisions (1). ϭ was used to estimate the telomere length in base pair: bp molecules of To study the role of CDK inhibitors during in vitro culture of equivalent soluble fluorochrome ϫ 0.495 (33). CD8ϩ T lymphocytes from the time of their isolation from human blood, up to the point when they senesced, we analyzed the levels Results Kip1 Expression of p16 protein does not coincide with replicative of p16 and p27 (hereafter p27) proteins on Western blots (Fig. senescence of CD8ϩ T lymphocytes cultured in vitro 1B). Data obtained from two independent experiments showed a ϩ progressive increase of p16 protein levels during culture, with a Human CD8 T lymphocytes isolated from peripheral blood are peak after ϳ20 PD. Surprisingly, this did not coincide with senes- predominantly small quiescent cells in the G0 phase of the cell cence of these cells. Instead, p16 expression had decreased to very cycle. When subjected to stimulation with PHA and irradiated al- ϩ low levels by the time the cells became senescent. p16 was unde- logeneic PBMC, the CD8 T cells became large blasts and pro- tectable in freshly isolated CD8ϩ T cells and in cells rendered liferated, as reflected by the high proportion of cells in the S/G2/M quiescent by IL-2 starvation. In contrast, p27 levels were particu- phases of the cell cycle. During seven to eight consecutive rounds larly high in these two populations, clearly above the levels in ϳ of mitogenic stimulation over 80 days, these cells underwent 30 senescent cells. These results suggest that neither p16 nor p27 is PD until they reached a state in which net increase in the total cell responsible for the senescence of CD8ϩ T lymphocytes in vitro. number was no longer observed upon further stimulation (Fig. 1A). This state was previously described for lymphocytes and has been De novo expression of p16 is a delayed response of some T

deﬁned as replicative senescence by analogy with the term used to lymphocytes to mitogen stimulation Downloaded from To investigate the role of the transient increase in the levels of p16 during the proliferative life span of the CD8ϩ T cell population as a whole, we analyzed these populations by ﬂow cytometry after staining them intracellularly with anti-p16 and anti-pRb Abs. When we stained ex vivo isolated CD8ϩ T cells with CFSE before

stimulation and analyzed their response to mitogen by flow cy- http://www.jimmunol.org/ tometry, it became apparent that more than one-half of the cells failed to divide at all, whereas ϳ11% underwent a single division and then arrested. The remainder of the cells divided from two to at least seven times during the 7-day culture period (Fig. 2A; data not shown). When we combined CFSE staining with intracellular staining for p16, we found (although not detectable by Western blotting) a low level of p16 in freshly isolated CD8ϩ T cells (day 0; Fig. 2B). Four days after mitogenic stimulation, p16 was undetectable, but by guest on September 27, 2021 after a further 3 days it was again detectable to levels comparable to that in unstimulated cell. Because at this time point ϳ99% of the population was made up of cells that had undergone several divisions, most of p16 in these cells must have been synthesized after day 4. To define more precisely the dynamics of p16 expression, we determined the levels of this protein in cells that have undergone different number of divisions according to their CFSE concentration. A representative example of histograms obtained 4 days after stimulation is depicted in Fig. 2C. High levels of p16 were found in cells that did not divide in response to stimulation. In contrast, p16 was barely detectable in cells that had undergone at least four divisions, indicating that the apparent decrease in p16 expression in the bulk population 4 days after stimulation is the result and increase in proliferating, p16-negative cells. FIGURE 1. The p16, p27, and pRb expression patterns differ in proliferating, quiescent, and senescent lymphocytes. A, Growth kinetics (PD vs High proportions of cycling T lymphocytes accumulate p16 ϩ time) of a CD8 T lymphocyte population cultured by repeated stimulation during the successive rounds of mitogen stimulation cycles. PD was calculated by periodic cell counting. B, Evolution along the culture of the protein content; cell cycle distribution; apoptosis index; and The findings described above were made with cells obtained dur- ϩ expression of p27, p16, and all of the pRb family proteins. To compare ing the first cycle of mitogenic stimulation of CD8 T cells in protein levels per cell at different time points, extracts from equal numbers vitro. When we determined p16 expression on days 4, 7, and 10 of of cells (8 ϫ 105 cells) were analyzed on Western blots. These analyses each of the following seven stimulation cycles before the cells were performed on ex vivo isolated cells (lanes 1 and 2); mitogen-activated senesced, we observed a similar pattern in each cycle (Fig. 3A). cells following 7, 14, 28, 35, and 56 days of culture (lanes 3–7 and 9); The percentage of p16-containing cells declined during the first 4 senescent cells (lane 8); and IL-2-starved cells (lane 10). Lane 1, Shows days after stimulation (see day 4, Fig. 3A), due to an increase in the signals obtained either after a maximum exposition (for pRb) or with an p16-negative proliferating cells (see day 4, Fig. 3B). As observed amount of protein extract corresponding to 3.2 ϫ 106 instead of 8 ϫ 105 cells (for p16 and p27). In the latter case, this allows the comparison be- during the first cycle of stimulation, the percentage of p16-express- tween ex vivo nondividing cells with in vitro activated cycling cells based ing cells began to increase from day 5 onward. This augmentation on the amount of loaded protein. Images obtained from two different ex- was particularly stark at days 7 and 10 after stimulation. The size position times of the same blot are shown for p107 and p130. Œ, Indicate of the fraction of p16-positive cells increased after each consecu- the time point at which the cells were harvested for further analysis. tive mitogenic stimulation up to the fourth to fifth stimulation (Fig. 3338 p16INK4a EXPRESSION IN CULTURED HUMAN CD8ϩ T LYMPHOCYTES

FIGURE 2. Expression of p16 is related to the proliferative state of T lymphocytes after in vitro stimulation. Ex vivo isolated CD8ϩ T lymphocytes were stained with CFSE and stimulated, and cell samples collected at days 0, 3, 4, 5, and 7 were analyzed for p16 expression. A, CFSE fluorescence showing Downloaded from the peaks corresponding to cells that underwent the indicated number of cell divisions. A 10-fold image amplification of the CFSE analysis (corresponding to the highest fluorescence peaks) is depicted at day 7. B, p16 expression on the T cell populations after 0, 4, and 7 days of culture. Shaded and open histograms represent the fluorescence obtained with, respectively, p16 mAb and its corresponding isotype control. C, Analysis of p16 expression at day 4 following stimulation, after gating the cells according to their CFSE concentration (as indicated by the number of cell division). D, Analysis of p16 expression in U2OS (p16Ϫ) and HeLa (p16ϩ) cell lines following intracellular staining using anti-p16 mononuclear Ab. HeLa cells expressed high levels of the p16 protein (180), whereas virtually no U2OS cells were stained positive for p16 (Ͻ0.1). Note that the p16 histogram profile for U2OS cells fully http://www.jimmunol.org/ overlaps that of the isotype control. All depicted values (B–D) represent the normalized mean fluorescence for each respective Ab, as described in Materials and Methods. Comparable data were obtained from two additional independent experiments.

3A), so much so that in the fifth cycle 35% of the CFSElow cells which p16 was expressed as the result of de novo synthesis (see present on day 7 contained high amounts of p16 (see Fig. 3B). day 7; Fig. 4B). Because these cells are descendants of the cycling p16-negative cells present on day 4, their p16 expression can only be the result of de novo synthesis of this protein. From the sixth stimulation Transient knockdown of p16 expression by specific shRNA by guest on September 27, 2021 onward, the fraction of p16-positive cells present on days 7 and 10 resulted in increased proliferation of T lymphocytes after stimulation decreased, and at senescence (after 85 days of To further investigate the causal link between activation-induced culture) only a very small fraction of senescent cells expressed p16 expression and the proliferative potential of mitogen-activated p16, and those that do expressed very low levels of it. The p16 CD8ϩ T lymphocytes, we knocked down p16 gene expression expression did not significantly change after the ninth cycle, or with two lentiviral vectors, each expressing a different p16-specific when the cells were kept in culture for an additional period of time shRNA (p16shRNA-A or p16shRNA-B) (26, 27). In vitro cultured without stimulation (data not shown). T cells were transduced once with the lentiviral supernatant These results are consistent with the p16 levels in bulk popula- p16shRNA-A or p16shRNA-B. The ability of each specific tion of stimulated cells as measured by Western blotting (Fig. 1B). shRNA to knock down the expression of p16 protein was moni- Altogether, our data suggest that newly synthesized p16 limits the tored, as shown in Fig. 5A. Following p16 shRNA treatment, a ϩ proliferation of CD8 T lymphocytes that respond to mitogen and 2-fold reduction in the amount of p16 expression was observed in IL-2, but that it is not required for the loss of mitogen responsive- transduced T lymphocytes at day 4 after stimulation. However, ness called senescence. knockdown of p16 expression was only transient, as there was no significant difference in p16 levels between transduced and control T lymphocytes expressing high levels of p16 are arrested in G0/ T cells from day 7 onward (data not shown). We next assessed the G1 phase of the cell cycle number of PDs (Fig. 5B) as well as the proportion of cells in the

The expression of p16 has been associated, in epithelial cells, with S/G2/M phases of cell cycle (Fig. 5C). Transient reduction of p16 irreversible growth arrest (33, 34). Using BrdU incorporation as- gene expression resulted in a significant increase of the numerical says, we next investigated the consequence of p16 expression dur- expansion of transduced cells from day 4 to 7 following mitogenic ing cell cycle replication of CD8ϩ T lymphocytes on days 4 and 7 stimulation in comparison with control cells. Moreover, this net after mitogenic stimulation (Fig. 4). Almost all T cells that had growth advantage was accompanied by a higher proportion of cy- incorporated BrdU and progressed through the cell cycle were also cling cells within the fraction of transduced GFP-positive cells p16 negative. In contrast, a significant proportion of cells that when compared with the one present among GFP-negative cells failed to divide in response to stimulation and were thus BrdU (53 vs 38%; Fig. 5C). Although the p16 knockdown appears at first negative expressed the p16 protein (Fig. 4A). These p16-positive T flush to be disappointingly transient, in a roundabout way, it has cells were found exclusively within the G0/G1 phase of the cell actually created a quasi p16 off-on situation, which is closely par- cycle, as assessed by 7-AAD costaining of the DNA content (Fig. alleled by a transient boost in PD of the cells. Hence, the tran- 4B). Our data indicate that the accumulation of p16 may be re- sientness of the p16 knockdown has inadvertently lent even more sponsible in preventing a fraction of cells from proliferating weight to the conclusion that reduction of p16 level increases when stimulated. This is also true for CD8ϩ T lymphocytes in CD8ϩ T cell expansion, and re-establishment of the p16 levels The Journal of Immunology 3339

FIGURE 4. CD8ϩ T lymphocytes expressing p16 are arrested in the

G0/G1 phase of the cell cycle. Mitogen-stimulated T lymphocytes (third cycle) were incubated during1hinthepresence of BrdU. A, Representa- tive dot plot of combined p16 and BrdU intracellular staining at day 4 after stimulation. B, BrdU incorporation and DNA content (7-AAD; linear scale) analysis on gated p16-negative (R1) and p16-positive (R2) T cells at days

4 and 7. The proportion (in %) of cells, respectively, in G0/G1 (see arrow; Ϫ ϩ BrdU ) and in S/G2/M (BrdU ) phases of cell cycle is depicted. Downloaded from (31, 32). As shown in Fig. 6A, a strong decline in telomere fluorescence was observed between the third and fifth cycles of stimulation, corresponding to a loss of ϳ3.7-kb telomeric DNA. How- ever, from the fifth stimulation onward until replicative senescence telomere fluorescence remained stable and corresponded to ϳ5.5

kb (Fig. 6A). In contrast, human T cell clones that senesced after http://www.jimmunol.org/ cultivation by serial recloning for extended periods of time (30) displayed extremely short telomeres (2 kb; see control clones, Fig. 6A). This comparison indicates that telomere attrition is unlikely to be involved in the replicative senescence of bulk population of CD8ϩ T cells, in agreement with previous reports (35). Intrigu- ingly, these results do not corroborate those obtained from senescent T cells that were subjected to consecutive rounds of recloning (30). To date, we do not have a rational explanation for the ap- parently contradictory findings concerning the role of telomere by guest on September 27, 2021 shortening in the senescence of CD8ϩ T cells cultured in a different manner. Our data suggest that induction of p16 expression in bulk cultures of CD8ϩ T cells is independent of telomere attrition. Further ϩ FIGURE 3. The p16 expression kinetics in CD8 T cell populations evidence for this comes from the analysis of p16 in CD8ϩ T cell ϩ following repeated rounds of mitogen stimulation. A, CD8 T cells were clones that ectopically express the human telomerase catalytic sub- cultivated following eight cycles of stimulation until they senesced. Levels unit (hTERT) (30). These hTERT-transduced T clones express of p16 expression were measured 0, 4, 7, and 10 days after each cycle of high levels of telomerase and have elongated their telomeres up to stimulation, and the percentage of p16-positive cells was calculated, as described in Materials and Methods. B, Representative dot plots and his- 13-kb average length (see control cells, Fig. 6A). Their replicative tograms of combined p16 and CFSE analysis were obtained from samples life span exceeds 200 PD compared with 108 PD in control clones. at days 4 and 7 after the fifth cycle of stimulation. The percentage of Nevertheless, a significant fraction of p16-positive cells was gen- p16-positive cells in histograms was derived after gating in the highest (H, erated as a consequence of mitogen stimulation of T cell clones CFSEhigh) or the lowest (L, CFSElow) CFSE fluorescence regions from transduced with either control or hTERT vectors (Fig. 6B). As in p16/CFSE dot plots. primary CD8ϩ T cell bulk cultures, p16 expression increased after 7 days of stimulation to comparable levels (Fig. 3A). Taken together, these results indicate that mitogen-induced p16 expression obliterates this increase. Together, our results show that activation- can be mediated by a mechanism that is independent of telomere induced p16 limits the numerical expansion of mitogen-activated T length. lymphocytes in vitro. The active form of pRb is predominant in senescent CD8ϩ T Activation-induced p16 expression is not a result of telomere lymphocytes attrition We observed irreversible growth arrest or replicative senescence in One question that our findings raise is what event or stimulus is our bulk culture system after seven to eight consecutive rounds of responsible for the onset of p16 synthesis during a stimulation mitogenic stimulation during ϳ80 days of culture (Fig. 1A). Care- cycle. In cultured cells, p16 has been described as a mediator of ful analysis using CFSE staining revealed that 99.9% of the se- stress signals due to suboptimal culture conditions (9), but its ex- nescing CD8ϩ T cells failed to proliferate in response to mitogenic pression can be also induced by the presence of critically short stimulation (PHA) or when challenged with PMA and ionomycin telomeres (19). To explore a possible relationship between p16 (our unpublished data). Our findings that the senescing cells con- expression and telomere loss in in vitro cultured CD8ϩ T cells, we tained no or very low levels of p16 and had telomeres the average monitored the average length of telomere repeats using flow FISH length of which considerably exceeded that of senescing clones 3340 p16INK4a EXPRESSION IN CULTURED HUMAN CD8ϩ T LYMPHOCYTES

FIGURE 5. shRNA-mediated p16 knockdown increases proliferation of human T lymphocytes. In vitro cultivated T cells (second or third cycle of stimulation) were transduced once with p16shRNA-A or p16shRNA-B len- FIGURE 6. Relationship between p16 expression and telomere attrition tiviral vector. Experiments on untransduced cells (control) as well as on in long-term culture of CD8ϩ T lymphocytes and in telomerase-expressing Downloaded from cells transduced with the empty vector were performed in parallel. Mito- T cell clones. A, Telomere ﬂuorescence analysis in CD8ϩ T lymphocytes gen-stimulated T lymphocytes were analyzed between days 3 and 8 for p16 cultivated after third, ﬁfth, sixth, and seventh rounds of mitogen stimula- expression by intracellular staining (A; proportion of p16-positive cells tion. Histograms obtained from two control T cell clones with either long relative to the control cells), their numerical expansion by cell counting (13 kb) or very short telomere lengths (2 kb) are depicted. The dotted line

(B), and their proportion in S/G2/M phases of cell cycle by BrdU incor- was arbitrarily set at the mean telomere fluorescence obtained from the fifth poration (C; day 4). Data collected from two independent experiments stimulation and allows the direct telomere fluorescence comparison be- (Expts. 1 and 2) are depicted. Because each experiment includes CD8 T tween samples. B, Characterization of p16 protein in CD8ϩ T cell clones http://www.jimmunol.org/ lymphocytes isolated from two different healthy donors, data are expressed transduced with a retroviral vector containing GFP or GFP/hTERT cDNAs. as mean Ϯ SD. Control cells; E, p16shRNA-A-transduced cells; F, These clones were obtained and cultured following serial rounds of sub- p16shRNA-B-transduced cells; gray circles. cloning steps (30). The percentages of p16-positive cells were monitored in T cell clones from the indicated subcloning steps (II, VIII, and IX) at days 4 and 7 after mitogen stimulation. Both isotype control (open) and p16 indicate that senescence in the bulk-cultured CD8ϩ T cells was (gray) histograms are depicted. triggered neither by telomere loss (Fig. 6A) nor by p16 expression (Figs. 1B and 3A). Previous reports have shown that human fibroblasts and epithe- with the observations made with other human cell types and sug- by guest on September 27, 2021 lial cells express high levels of the hypophosphorylated, active gest that a high ratio of active vs total pRb is associated also with form of pRb protein at senescence (36). These and other experi- lymphocyte senescence. What is unclear, at present, is what con- mental evidences support the notion that pRb pathway plays a trols pRb phosphorylation in these cells. It does not appear to crucial role in the entry and/or the maintenance of senescence in be p16. various human cell types (reviewed in Ref. 37). To characterize the state of the pRb protein during the different cycles of stimulation Discussion in T lymphocytes, we analyzed the phosphorylation patterns of the In vitro expansion of human cytolytic CD8ϩ T lymphocytes by pRb protein family, p107, p130, and pRb, in these cells on Western repeated mitogenic stimulation is widely used for clinical and re- blots (Fig. 1B). The pRb protein, which existed predominantly in search purposes. Adoptive immunotherapy of cancer has been lim- the phosphorylated inactive form in the proliferating T cells, was ited by the fact that under currently used tissue culture conditions, found to be in the hypophosphorylated active form in senescent tumor-infiltrating lymphocyte populations or tumor-specific CTL cells. This suggests that in senescent CD8ϩ T lymphocytes, pRb clones can be expanded at most to 109-1011 cells. As reinfused T remains in the active state independently of p16. Although prolif- cells often do not persist longer than a few days in the host, the erating lymphocytes contained high levels of the inactive forms of efficiency of adoptive immunotherapy might be enhanced if culture the pRb-related proteins p107 and p130, only very low levels of methods that allow the generation in vitro of sufficient numbers of their active forms were found at senescence. CTLs for multiple reinfusions into patients can be developed (2). We also measured the level of expression of total pRb and active This complex task would be made easier by a better understanding pRb by intracellular staining (38). The evolution of pRb expression of the molecular mechanisms that limit the proliferative potential was similar in all rounds of stimulation. A typical example is de- of in vitro cultured CTL. picted in Fig. 7 (third stimulation). In general, actively proliferat- The culture system used in the experiments described in this ing cells (day 7) showed high levels of total pRb protein. The work allows bulk CD8ϩ T lymphocytes to undergo ϳ30 PD in absolute levels of active pRb were moderately higher than those in eight rounds of stimulation with PHA and IL-2. This means that in nondividing quiescent cells (see ϩday 22, Fig. 7) but the active/ our culture conditions we can produce 109 cells per initial T cell. total pRb ratio was significantly lower, indicating the presence of However, analysis by CFSE staining showed that a fraction of the high levels of cycling-dependent kinase activity in these cells. In cells undergoes at least eight divisions during one stimulation cy- agreement with the results obtained by Western blots (Fig. 1B), cle. If all of the cells divided eight times per stimulation, then we cells cultured for 85 days that enter replicative senescence showed would obtain 2 ϫ 1019 descendants from each initial cell! Under- a high ratio of active/total pRb. Senescent cells cultured for an standing the extrinsic and intrinsic factors that prevent a T cell additional 10 days possessed low levels of total pRb mostly found from realizing this maximum proliferation potential of eight divi- in the active form (ratio ϭ 0.95; Fig. 7). Our results are consistent sions per cycle should provide us with clues as to how we can The Journal of Immunology 3341

tem. Although the levels of p16 in the former were not measured, it would not at all be surprising if p16 was also exerting a limit to their system of expansion. What causes a fraction of mitogen-stimulated cells to express p16 after stimulation? The average length of telomeres in these populations makes it unlikely that p16 expression is triggered by a disruption of telomeric structures. The ﬁnding that hTERT-transduced T cell clones with long telomeres also express increased FIGURE 7. pRb expression correlates with the proliferative state of T amount of p16 following mitogen stimulation (Fig. 6) reinforces lymphocytes, and senescent cells express high levels of the active form of this conclusion. There is some evidence that p16 appearance can pRb. Total and hypophosphorylated (active) pRb expression were mea- be induced by stress due to inappropriate culture conditions (9); sured by intracellular staining in CD8ϩ T populations at days 4, 7, 10, and the resulting growth arrest is referred to as culture-induced senes- 22 after the third cycle of stimulation (day 20), as well as at replicative cence or culture shock (40). It is possible that inadequate culture senescence (day 85) and after additional 10 days (day 95). Both isotype conditions contribute to the appearance of p16 in bulk-cultured T control (open) and total or active pRb (gray) histograms are shown. The cells. However, it is intriguing that although all cells were grown normalized mean ﬂuorescence for each histogram and the active/total pRb under the same conditions, only a fraction of them expressed p16. ratio were calculated, as described in Materials and Methods. Should culture shock be the cause of p16 expression, it would imply that the culture conditions were inappropriate for some cells

while being appropriate for others. Moreover, one would expect a Downloaded from improve culture conditions to increase the fraction of cells that gradual and linear increase of p16 expression following each cycle proliferate maximally. In the present study, we describe that there of stimulation with high levels of p16 at replicative senescence. are at least two mechanisms that lead to irreversible growth arrest Instead, we found that from the sixth round of stimulation onward, ϩ of bulk-cultured CD8 T lymphocytes. the amount of p16-positive cells significantly diminished and senescent T cells expressed extremely low to undetectable levels of Activation-induced p16 expression limits the in vitro expansion ϩ p16 (Figs. 1 and 3). It may be suggested as another view that the http://www.jimmunol.org/ of human CD8 T lymphocytes expression of p16 in a fraction of bulk T cells could be the result The first mechanism is characterized by the appearance of p16 of an intrinsic mechanism that regulates the number of times that protein in a subset of cells during each cycle of stimulation. Our these cells can divide. The notion of a physiological role for p16 data indicate that the cells that are induced to synthesize p16 in a in regulating the proliferative potential of human T lymphocytes is given cycle will not proliferate in response to the next mitogen consistent with recent observations that will be discussed in the stimulation. Thus, during the early phase of each cycle, the p16- next section. positive cells were diluted out by proliferating p16-negative cells, a fraction of which subsequently expressed p16. During the early Does p16 play a physiological role in regulating the cycles, this fraction was small, but it gradually increased and proliferative potential of human T lymphocytes? by guest on September 27, 2021 reached a maximum during the fifth round of stimulation when As in keratinocytes (41) or fibroblasts (16, 17), p16 expression 35% of the proliferating population was p16 positive at the end of cannot be detected in freshly isolated ex vivo CD8ϩ T lympho- the cycle (Fig. 3). cytes by Western blot analysis (Fig. 1B). However, by using in- Our observations reveal several important points. First, others tracellular staining techniques, we have found low, but readily de- have reported p16 accumulation in long-term culture of fibroblasts tectable levels of p16 in the latter cells (Fig. 2B), a result that is (6, 17, 18). We demonstrate that in cultured lymphocytes this is not consistent with a recent comparative microarray analysis that re- a process that uniformly occurs in all cells, but that de novo p16 vealed that p16 mRNA levels were 2 times higher in freshly iso- appears only in a fraction of T cells that had been actively cycling lated human T cells than in the same population 3 days after in (Fig. 3). Moreover, activation-induced p16 expression is not re- vitro activation (42). These observations suggest a physiological lated to activation-induced cell death and occurs in a different cell role for p16 in CD8ϩ T lymphocytes, in line with the thymic subpopulation (data not shown). Second, this subset of p16-ex- hyperplasia in mice lacking p16 (24). That p16 indeed regulates pressing cells is found exclusively within the G0/G1 phase of the the proliferative potential of T cells in vivo is further suggested by cell cycle (Fig. 4). Third, in line with these results, transient knock- the elevated p16 RNA levels in the differentiated subset of periph- ing down of p16 expression led to the concomitant increase in the eral blood T cells (43). proportion of actively cycling cells (Fig. 5). Thus, our data clearly The entry into or exit of CD8ϩ T lymphocytes from the cell indicate that accumulating p16 is responsible for the exit of a frac- cycle may be determined not merely by the levels of CDK inhib- tion of cells from the proliferating population following each stim- itors such as p16, but also by the levels of the CDKs themselves. ulation. These results also suggest that the number of divisions that Indeed, Veiga-Fernandes and Rocha (44) recently reported that a cell will undergo after stimulation is determined by the onset of elevated levels of active CDK6 in mouse CD8 memory T lym- de novo synthesis of p16. The earlier synthesis starts the fewer phocytes favor rapid cell division. Another important parameter divisions the cell can make. Such a process would whittle the involves the subtype of CD8ϩ T lymphocytes, as bulk T cells can number of proliferating cells down after each cycle, and make it be phenotypically and functionally divided into naive, memory, difficult to achieve eight PD per stimulation. Our observations are and effector subsets. For instance, it is possible that memory T in agreement with a recent report showing that T lymphocytes cells are maintained by high levels of CDK6 in a preactivated from p16 knockout mice proliferate more than cells from wild-type stage, allowing them to progress more rapidly through the cell animals in response to anti-CD3 plus anti-CD28 (24). Recently, cycle than naive cells (44). Similarly, human memory T cells do Maus et al. (39) demonstrated that artificial APCs expressing li- also present different proliferation potentials in response to homeo- gands for the TCR, CD28, and 4-1BB enhance the expansion of T static cytokines when compared with other subsets (45). In that cells in vitro. Interestingly, the efficiency of this system in ampli- context, single cell analysis of naive, memory, and effector CD8ϩ fying T cell numbers appears to be comparable to that of our sys- T lymphocyte subpopulations will reveal whether levels of p16 or 3342 p16INK4a EXPRESSION IN CULTURED HUMAN CD8ϩ T LYMPHOCYTES

CDK6 expression can be differentially regulated in those subsets vivo. Further work is needed to elucidate completely the signals and may reﬂect potential differences in their in vivo and in vitro and pathways that limit T lymphocyte proliferation in vivo and in proliferative capacity. vitro. A better understanding of the factors that restrict T cell ex-

ϩ pansion in vitro will contribute to the design of standard culture Replicative senescence of CD8 T lymphocytes after extended in conditions that make efficient production of these cells as a ther- vitro culture is independent of p16 apeutic tool against infections or cancer possible. In the bulk cultures of CD8ϩ T cells used in this study, p16 limited the fraction of cells that responded to the next cycle of stimulation, Acknowledgments but the fraction of p16-positive cells never exceeded 35% and We are thankful to Drs. Markus Nabholz and Joachim Lingner for critically diminished after the fifth cycle. Nevertheless, eventually the cul- reading the manuscript, and to Dr. Claus Azzalin for his help with the ture reached a stage at which Ͻ0.1% of the cells divided in re- H2AX staining. We thank Patricia Corthe´sy-Henrioud and sponse to mitogenic stimulation (Fig. 3; day 85, eighth cycle of Se´verine Reynard for excellent technical assistance. stimulation). In analogy to a similar phenomenon in fibroblasts (46, 47), we refer to this permanent growth arrest as replicative Disclosures senescence. Senescence is thought to be a protective mechanism The authors have no financial conflict of interest. against the development of tumors, and in fibroblasts, is mediated References by the activation of DNA damage signaling (p53 and pRb) path- 1. Effros, R. B., and G. Pawelec. 1997. Replicative senescence of T cells: does the ways triggered by critically short telomeres (48, 49). Like senes- Hayflick Limit lead to immune exhaustion? Immunol. Today 18:450. ϩ cent fibroblasts (36), senescent T lymphocytes expressed moderate 2. Ho, W. Y., C. Yee, and P. D. Greenberg. 2002. Adoptive therapy with CD8 T Downloaded from levels of pRb, most of which were in the hypophosphorylated ac- cells: it may get by with a little help from its friends. J. Clin. Invest. 110:1415. 3. Muller, H., and K. Helin. 2000. The E2F transcription factors: key regulators of tive form (Figs. 1B and 7). But, unlike some strains of senescent cell proliferation. Biochim. Biophys. Acta 1470:M1. fibroblasts or epithelial cells (16, 17, 41, 50), senescent bulk-cul- 4. Grana, X., J. Garriga, and X. Mayol. 1998. Role of the retinoblastoma protein ϩ family, pRB, p107 and p130 in the negative control of cell growth. Oncogene tured CD8 T cells express almost no p16 protein. Thus, senes- 17:3365 ϩ . cence of CD8 T cell bulk cultures does not appear to be mediated 5. Takahashi, Y., J. B. Rayman, and B. D. Dynlacht. 2000. Analysis of promoter binding by the E2F and pRB families in vivo: distinct E2F proteins mediate by p16. It occurs at a stage when the average telomere length has http://www.jimmunol.org/ activation and repression. Genes Dev. 14:804. not yet been reduced to the level at which critically short telomeres 6. Allsopp, R. C., H. Vaziri, C. Patterson, S. Goldstein, E. V. Younglai, are expected to induce senescence, and we have previously re- A. B. Futcher, C. W. Greider, and C. B. Harley. 1992. Telomere length predicts ported that extension of telomeres by ectopic expression of telom- replicative capacity of human fibroblasts. Proc. Natl. Acad. Sci. USA 89:10114. 7. Bodnar, A. G., M. Ouellette, M. Frolkis, S. E. Holt, C. P. Chiu, G. B. Morin, erase does not prevent such cultures from becoming senescent C. B. Harley, J. W. Shay, S. Lichtsteiner, and W. E. Wright. 1998. Extension of (35). Moreover, it has been recently shown that several compo- life-span by introduction of telomerase into normal human cells. Science 279:349. nents of the DNA damage response machinery, including phos- 8. Vaziri, H., and S. Benchimol. 1998. Reconstitution of telomerase activity in phorylated H2AX, 53bp1, and NBS1, form foci at dysfunctional normal human cells leads to elongation of telomeres and extended replicative life telomeres in senescent fibroblasts (48). In contrast, we have found span. Curr. Biol. 8:279.

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